scholarly journals Genetic engineering of Pseudomonas chlororaphis Lzh-T5 to enhance production of trans-2,3-dihydro-3-hydroxyanthranilic acid

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kaiquan Liu ◽  
Ling Li ◽  
Wentao Yao ◽  
Wei Wang ◽  
Yujie Huang ◽  
...  
Keyword(s):  
Shikimate Pathway ◽  
Pentose Phosphate ◽  
Genetically Engineered ◽  
Regulatory Genes ◽  
Pseudomonas Chlororaphis ◽  
Chiral Materials ◽  
Key Genes ◽  
Phenazine Production ◽  
Hydroxyanthranilic Acid ◽  
Natural Peptides

AbstractTrans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) is a cyclic β-amino acid used for the synthesis of non-natural peptides and chiral materials. And it is an intermediate product of phenazine production in Pseudomonas spp. Lzh-T5 is a P. chlororaphis strain isolated from tomato rhizosphere found in China. It can synthesize three antifungal phenazine compounds. Disruption the phzF gene of P. chlororaphis Lzh-T5 results in DHHA accumulation. Several strategies were used to improve production of DHHA: enhancing the shikimate pathway by overexpression, knocking out negative regulatory genes, and adding metal ions to the medium. In this study, three regulatory genes (psrA, pykF, and rpeA) were disrupted in the genome of P. chlororaphis Lzh-T5, yielding 5.52 g/L of DHHA. When six key genes selected from the shikimate, pentose phosphate, and gluconeogenesis pathways were overexpressed, the yield of DHHA increased to 7.89 g/L. Lastly, a different concentration of Fe3+ was added to the medium for DHHA fermentation. This genetically engineered strain increased the DHHA production to 10.45 g/L. According to our result, P. chlororaphis Lzh-T5 could be modified as a microbial factory to produce DHHA. This study laid a good foundation for the future industrial production and application of DHHA.

scholarly journals Genetic Engineering of Pseudomonas Chlororaphis Lzh-T5 to Enhance Production of Trans-2,3-Dihydro-3-Hydroxyanthranilic Acid

2020 ◽  
Author(s):  
Ling Li ◽  
Zhenghua Li ◽  
Xuehong Zhang ◽  
Wei Wang ◽  
Yujie Huang ◽  
...  
Keyword(s):  
Metal Ions ◽  
Shikimate Pathway ◽  
Pentose Phosphate ◽  
Genetically Engineered ◽  
Regulatory Genes ◽  
Pseudomonas Chlororaphis ◽  
Key Genes ◽  
Phenazine Production ◽  
Hydroxyanthranilic Acid ◽  
Natural Peptides

Abstract Background: Trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) is a cyclic β-amino acid used for the synthesis of non-natural peptides and chiral materials. It is an intermediate product of phenazine production in Pseudomonas spp . Lzh-T5 is a P. chlororaphis strain isolated from tomato rhizosphere found in China. It can synthesize three antifungal phenazine compounds. Results: Disrupting the phzF gene of P. chlororaphis Lzh-T5 results in DHHA accumulation. Several strategies were used to improve production of DHHA: enhancing the shikimate pathway by overexpression, knocking out negative regulatory genes, and adding metal ions to the medium. In this study, three regulatory genes ( psrA , pykF, and rpeA ) were-disrupted in the genome of P. chlororaphis Lzh-T5, yielding 4.55 g/L of DHHA. When six key genes selected from the shikimate, pentose phosphate, and gluconeogenesis pathways were overexpressed, the yield of DHHA increased to 6.89g/L. Fe 3+ was added to the medium for DHHA fermentation. This genetically engineered strain increased the DHHA production to 10.45g/L. Conclusions: P. chlororaphis Lzh-T5 could be modified as a microbial factory to produce DHHA by inactivating phzF , disrupting negative regulatory genes, overexpressing key genes, and adding metal ions to medium for fermentation.

scholarly journals Polyhydroxyalkanoate (PHA) Polymer Accumulation and pha Gene Expression in Phenazine (phz-) and Pyrrolnitrin (prn-) Defective Mutants of Pseudomonas chlororaphis PA23

Polymers ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 1203 ◽  
Author(s):  
Parveen Sharma ◽  
Riffat Munir ◽  
Jocelyn Plouffe ◽  
Nidhi Shah ◽  
Teresa Kievit ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reducing Power ◽  
Pentose Phosphate ◽  
Pha Synthase ◽  
Pseudomonas Chlororaphis ◽  
Glucose Medium ◽  
Wild Type ◽  
Pha Production ◽  
Phenazine Production ◽  
Altered Gene

Pseudomonas chlororaphis PA23 was isolated from the rhizosphere of soybeans and identified as a biocontrol bacterium against Sclerotinia sclerotiorum, a fungal plant pathogen. This bacterium produces a number of secondary metabolites, including phenazine-1-carboxylic acid, 2-hydroxyphenazine, pyrrolnitrin (PRN), hydrogen cyanide, proteases, lipases and siderophores. It also synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds under nutrient-limited conditions. Pseudomonads like P. chlororaphis metabolize glucose via the Entner-Doudoroff and Pentose Phosphate pathways, which provide precursors for phenazine production. Mutants defective in phenazine (PHZ; PA23-63), PRN (PA23-8), or both (PA23-63-1) accumulated higher concentrations of PHAs than the wild-type strain (PA23) when cultured in Ramsay’s Minimal Medium with glucose or octanoic acid as the carbon source. Expression levels of six pha genes, phaC1, phaZ, phaC2, phaD, phaF, and phaI, were compared with wild type PA23 by quantitative real time polymerase chain reaction (qPCR). The qPCR studies indicated that there was no change in levels of transcription of the PHA synthase genes phaC1 and phaC2 in the phz- (PA23-63) and phz- prn- (PA23-63-1) mutants in glucose medium. There was a significant increase in expression of phaC2 in octanoate medium. Transcription of phaD, phaF and phaI increased significantly in the phz- prn- (PA23-63-1) mutant. Mutations in regulatory genes like gacS, rpoS, and relA/spoT, which affect PHZ and PRN production, also resulted in altered gene expression. The expression of phaC1, phaC2, phaF, and phaI genes was down-regulated significantly in gacS and rpoS mutants. Thus, it appears that PHZ, PRN, and PHA production is regulated by common mechanisms. Higher PHA production in the phz- (PA23-63), prn- (PA23-8), and phz- prn- (PA23-63-1) mutants in octanoic medium could be correlated with higher expression of phaC2. Further, the greater PHA production observed in the phz- and prn- mutants was not due to increased transcription of PHA synthase genes in glucose medium, but due to more accessibility of carbon substrates and reducing power, which were otherwise used for the synthesis of PHZ and PRN.

scholarly journals Enhanced biosynthesis of arbutin by engineering shikimate pathway in Pseudomonas chlororaphis P3

2018 ◽  
Vol 17 (1) ◽  
Author(s):  
Songwei Wang ◽  
Cong Fu ◽  
Muhammad Bilal ◽  
Hongbo Hu ◽  
Wei Wang ◽  
...  
Keyword(s):  
Shikimate Pathway ◽  
Pseudomonas Chlororaphis

scholarly journals Role of the phenazine-inducing protein Pip in stress resistance of Pseudomonas chlororaphis

Microbiology ◽  
2011 ◽  
Vol 157 (2) ◽  
pp. 398-407 ◽  
Author(s):  
Geneviève Girard ◽  
Sébastien Rigali
Keyword(s):  
Quorum Sensing ◽  
Virulence Factors ◽  
Stress Resistance ◽  
Antibiotic Production ◽  
Fusaric Acid ◽  
Significant Inhibition ◽  
Stress Factors ◽  
Pseudomonas Chlororaphis ◽  
Model Studies ◽  
Phenazine Production

The triggering of antibiotic production by various environmental stress molecules can be interpreted as bacteria's response to obtain increased fitness to putative danger, whereas the opposite situation – inhibition of antibiotic production – is more complicated to understand. Phenazines enable Pseudomonas species to eliminate competitors for rhizosphere colonization and are typical virulence factors used for model studies. In the present work, we have investigated the negative effect of subinhibitory concentrations of NaCl, fusaric acid and two antibiotics on quorum-sensing-controlled phenazine production by Pseudomonas chlororaphis. The selected stress factors inhibit phenazine synthesis despite sufficient cell density. Subsequently, we have identified connections between known genes of the phenazine-inducing cascade, including PsrA (Pseudomonas sigma regulator), RpoS (alternative sigma factor), Pip (phenazine inducing protein) and PhzI/PhzR (quorum-sensing system). Under all tested conditions, overexpression of Pip or PhzR restored phenazine production while overexpression of PsrA or RpoS did not. This forced restoration of phenazine production in strains overexpressing regulatory genes pip and phzR significantly impairs growth and stress resistance; this is particularly severe with pip overexpression. We suggest a novel physiological explanation for the inhibition of phenazine virulence factors in pseudomonas species responding to toxic compounds. We propose that switching off phenazine-1-carboxamide (PCN) synthesis by attenuating pip expression would favour processes required for survival. In our model, this ‘decision’ point for promoting PCN production or stress resistance is located downstream of rpoS and just above pip. However, a test with the stress factor rifampicin shows no significant inhibition of Pip production, suggesting that stress factors may also target other and so far unknown protagonists of the PCN signalling cascade.

The global regulator GacS regulates biofilm formation in Pseudomonas chlororaphis O6 differently with carbon source

2014 ◽  
Vol 60 (3) ◽  
pp. 133-138 ◽  
Author(s):  
Ji Soo Kim ◽  
Yong Hwan Kim ◽  
Ju Yeon Park ◽  
Anne J. Anderson ◽  
Young Cheol Kim
Keyword(s):  
Carbon Source ◽  
Biofilm Formation ◽  
Defined Medium ◽  
Root Surface ◽  
Gray Mold ◽  
Systemic Resistance ◽  
Pseudomonas Chlororaphis ◽  
Wild Type ◽  
Tomato Leaf Mold ◽  
Phenazine Production

An aggressive root colonizer, Pseudomonas chlororaphis O6 produces various secondary metabolites that impact plant health. The sensor kinase GacS is a key regulator of the expression of biocontrol-related traits. Biofilm formation is one such trait because of its role in root surface colonization. This paper focuses on the effects of carbon source on biofilm formation. In comparison with the wild type, a gacS mutant formed biofilms at a reduced level with sucrose as the major carbon source but at much higher level with mannitol in the defined medium. Biofilm formation by the gacS mutant occurred without phenazine production and in the absence of normal levels of acyl homoserine lactones, which promote biofilms with other pseudomonads. Colonization of tomato roots was similar for the wild type and gacS mutant, showing that any differences in biofilm formation in the rhizosphere were not of consequence under the tested conditions. The reduced ability of the gacS mutant to induce systemic resistance against tomato leaf mold and tomato gray mold was consistent with a lack of production of effectors, such as phenazines. These results demonstrated plasticity in biofilm formation and root colonization in the rhizosphere by a beneficial pseudomonad.

Galacto-oligosaccharide hydrolysis by genetically-engineered alpha-galactosidase-producing Pseudomonas chlororaphis strains

2018 ◽  
Vol 13 ◽  
pp. 213-218 ◽  
Author(s):  
Daniel K.Y. Solaiman ◽  
Richard D. Ashby ◽  
Kawalpreet K. Aneja ◽  
Nicole V. Crocker ◽  
Yanhong Liu
Keyword(s):  
Genetically Engineered ◽  
Pseudomonas Chlororaphis ◽  
Alpha Galactosidase ◽  
Oligosaccharide Hydrolysis

scholarly journals darR and darS are regulatory genes that modulate 2-hexyl, 5-propyl resorcinol transcription in Pseudomonas chlororaphis PCL1606

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2670-2680 ◽  
Author(s):  
Claudia E. Calderón ◽  
Víctor J. Carrión ◽  
Antonio de Vicente ◽  
Francisco M. Cazorla
Keyword(s):  
Regulatory Genes ◽  
Gene Arrangement ◽  
Gene Promoters ◽  
Pseudomonas Chlororaphis ◽  
Transcriptional Initiation ◽  
Polycistronic Mrna ◽  
Reverse Transcription Pcr ◽  
Genetic Features ◽  
Rapid Amplification ◽  
Cross Inhibition

Pseudomonas chlororaphis PCL1606 synthesizes the antifungal antibiotic 2-hexyl, 5-propyl resorcinol (HPR), which is crucial for the biocontrol of fungal soil-borne pathogens. The genetic basis for HPR production lies in the dar genes, which are directly involved in the biosynthesis of HPR. In the present study, we elucidated the genetic features of the dar genes. Reverse transcription PCR experiments revealed an independent organization of the dar genes, except for darBC, which was transcribed as a polycistronic mRNA. In silico analysis of each gene revealed putative promoters and terminator sequences, validating the proposed gene arrangement. Moreover, experiments utilizing 5′ rapid amplification of cDNA ends were used to determine the transcriptional initiation sites for the darA, darBC, darS and darR gene promoters, and subsequently to confirm the functionality of these regions. The results of quantitative real-time PCR experiments indicated that biosynthetic dar genes were not only modulated through the global regulator gacS, but also through darS and darR. The interplay between darS and darR revealed transcriptional cross-inhibition. However, these results also showed that other regulatory parameters play a role in HPR production, such as the environmental conditions and additional regulatory genes.

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